TDP-43 was recently identified as one of the major proteins that accumulate in inclusions in sporadic Amyotrophic Lateral Sclerosis (ALS) and in Fronto-temporal dementia with ubiquitin inclusions (FTLD- U). Abnormalities in TDP-43 biology are sufficient to cause neurodegenerative disease because mutations in TDP-43 are linked to familial ALS, which suggests that TDP-43 is linked directly to the disease process. The work in this grant proposal will build on our discovery that TDP-43 regulates the translational response to stress. Neurons respond to stresses, such as occur in neurodegenerative diseases, by entering a state of translational arrest. Translationally silenced mRNA is sequestered into inclusions termed stress granules (SG), which are composed of an mRNA and RNA binding proteins, such as TIA-1. Inclusions composed of SGs are intriguing because of their dynamic nature. The proteins that nucleate SGs have the ability to reversibly aggregate partly because they contain aggregation prone polyglutamine and prion domains. TDP-43 is also a RNA binding protein. Our research demonstrates that TDP-43 co-localizes with SGs, and is essential for SG formation. Disease related mutations in TDP-43 appear to enhance SG formation, and conversely, agents that inhibit SG formation rapidly disaggregate TDP-43 inclusions. Our data also suggests that translational inhibitors can suppress or even rapidly disperse TDP-43 inclusions. We hypothesize that SGs contribute to formation of TDP-43 inclusions, such as occur in FTLD-U and ALS, and that inhibiting SGs can inhibit formation of TDP-43 inclusions. Aim 1 of this proposal will move our work from cell lines to primary neurons. Our preliminary work has been performed in cell lines such as human BE-M17 neuroblastoma cells, but the regulation of SGs differs in neurons. We will determine the types of conditions that stimulate formation of TDP-43 inclusions in primary cortical and spinal motor neuron cultures. We will also determine how mutations in TDP-43 affect inclusion formation. Aim 2 will identify the structural domains of TDP-43 inclusion formation and suppression/dispersion of TDP-43 inclusions. This work will identify the conditions and domains on TDP-43 that one can target to develop therapeutic approaches that can suppress or reverse TDP-43 inclusions and possibly treat ALS and FTLD-U. PUBLIC HEALTH RELEVANCE: This project investigates TDP-43, which is one of the major proteins that accumulate in Amyotrophic Lateral Sclerosis (ALS) and in Fronto-temporal dementia with ubiquitin inclusions (FTLD-U). We will determine the types of conditions that stimulate aggregation of TDP-43 and novel methods to prevent or reverse TDP-43 aggregation.